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The Plant Journal (2005) 42, 270–282 doi: 10.1111/j.1365-313X.2005.02368.x Plant-specific mitotic targeting of RanGAP requires a functional WPP domain Sun Yong Jeong1, Annkatrin Rose1, Jomon Joseph2, Mary Dasso2 and Iris Meier1,* Plant Biotechnology Center and Department of Plant Cellular and Molecular Biology, The Ohio State University, Columbus, OH 43210, USA, and 2 Laboratory of Gene Regulation and Development, National Institute of Child Health and Development, Bethesda, MD 20892, USA 1 Received 2 November 2004; revised 16 December 2004; accepted 14 January 2005. * For correspondence (fax 614 292 5379; e-mail [email protected]). Summary The small GTPase Ran is involved in nucleocytoplasmic transport, spindle formation, nuclear envelope (NE) formation, and cell-cycle control. In vertebrates, these functions are controlled by a three-dimensional gradient of Ran-GTP to Ran-GDP, established by the spatial separation of Ran GTPase-activating protein (RanGAP) and the Ran guanine nucleotide exchange factor RCC1. While this spatial separation is established by the NE during interphase, it is orchestrated during mitosis by association of RCC1 with the chromosomes and RanGAP with the spindle and kinetochores. SUMOylation of vertebrate RanGAP1 is required for NE, spindle, and centromere association. Arabidopsis RanGAP1 (AtRanGAP1) lacks the SUMOylated C-terminal domain of vertebrate RanGAP, but contains a plant-specific N-terminal domain (WPP domain), which is necessary and sufficient for its targeting to the NE in interphase. Here we show that the human and plant RanGAP-targeting domains are kingdom specific. AtRanGAP1 has a mitotic trafficking pattern uniquely different from that of vertebrate RanGAP, which includes targeting to the outward-growing rim of the cell plate. The WPP domain is necessary and sufficient for this targeting. Point mutations in conserved residues of the WPP domain also abolish targeting to the nuclear rim and the cell plate, suggesting that the same mechanism is involved in both targeting events. These results indicate that plant and animal RanGAPs undergo different migration patterns during cell division, which require their kingdom-specific targeting domains. Keywords: RanGAP, Ran cycle, nuclear envelope, mitosis, cell plate, plant. Introduction Ran is a small GTPase that in vertebrates has functions in nuclear transport, spindle formation, nuclear envelope (NE) re-assembly, and cell-cycle control (Arnaoutov and Dasso, 2003; Blow, 2003; Dasso, 2002; Li et al., 2003). RanGTP and Ran-GDP are interconverted by the activity of a cytosolic Ran GTPase activating protein (RanGAP) and the chromatin-bound nucleotide exchange factor RCC1. The spatial distribution of Ran-GTP and Ran-GDP, established by the spatial distribution of RanGAP and RCC1, is important for the different functions of Ran. During interphase, nuclear Ran is predominantly GTP-bound due to nuclear RCC1, and cytoplasmic Ran is predominantly GDPbound due to cytoplasmic RanGAP. This gradient across the NE is functional in the directionality of nuclear import and export, providing the information of compartment 270 identity for the appropriate loading and unloading of the transport factors (Görlich et al., 2003, and references therein). During vertebrate mitosis, Ran-GTP in the vicinity of the chromosomes displaces factors that promote microtubule polymerization from the inhibitory effect of bound importins, suggestive of a promoting role of Ran-GTP in spindle assembly (Gruss et al., 2001; Nachury et al., 2001; Wiese et al., 2001). During telophase, both the local accumulation of Ran-GTP and its hydrolysis have been implicated in the assembly of the daughter NEs (Hetzer et al., 2000; Zhang and Clarke, 2000). These findings indicate that the spatial positioning of RCC1 and RanGAP is equally important for the functions of Ran in mitotic cells and that these functions rely on the formation of a three-dimensional gradient between ª 2005 Blackwell Publishing Ltd Arabidopsis RanGAP1 at the cell plate 271 the two forms of Ran in the absence of a separating NE (Kalab et al., 2002). While vertebrate RCC1 remains chromatin-bound during cell cycle (reviewed in Dasso, 2002), RanGAP migrates from the NE to the vicinity of the spindle, with a sub-population observed at the kinetochores (Joseph et al., 2002; Matunis et al., 1996). In metazoans, RanGAP1 is conjugated with SUMO-1, a small ubiquitin-like protein. SUMOylation is required for NE association in interphase and for spindle and centromere association in metaphase (Joseph et al., 2002; Matunis et al., 1998). SUMOylated RanGAP1 binds to the nucleoporin RanBP2/Nup358 (Matunis et al., 1998). In metaphase cells, RanGAP1 and RanBP2/Nup358 co-localize in the vicinity of the spindle and the kinetochores, suggesting that the same protein–protein interaction is required for targeting of RanGAP1 to its interphase and its main mitotic location. RanGAP1 and RanBP2/Nup358 are targeted as a single complex which is both regulated by and essential for kinetochore–microtubule association (Joseph et al., 2004). Yeast homologs of RanGAP lack the C-terminal, SUMOylated targeting domain of mammalian RanGAPs and are not associated with the NE (Melchior et al., 1993). In contrast to higher animals and plants, the yeasts undergo closed mitosis without breakdown of the NE. Therefore, a purely or predominantly cytoplasmic location of yeast RanGAP would theoretically suffice for its function in nuclear import and export. Like vertebrate RanGAP – and unlike yeast RanGAP – plant RanGAP is associated with the NE during interphase (Rose and Meier, 2001). Arabidopsis RanGAP1 lacks the SUMOylated C-terminal domain of vertebrate RanGAP, but contains instead a plant-specific N-terminal domain, called WPP domain after a highly conserved tryptophan–proline– proline motif. The WPP domain is necessary and sufficient for targeting to the plant NE (Rose and Meier, 2001). Consistent with the presence of a different targeting domain, the Arabidopsis genome appears to lack a homolog of RanBP2/Nup358, suggesting that at least two modes exist for the spatial organization of the Ran cycle in higher eukaryotes. Knowledge about the similarities and differences in the mechanism of plant and animal RanGAP targeting (and, by extension, establishment of the Ran gradient) will be crucial to understand the degree of divergence between the two kingdoms. Here we show that the human and Arabidopsis RanGAP targeting domains are kingdom-specific. Unlike animal RanGAP, Arabidopsis RanGAP1 appears at the emerging cell plate in dividing plant cells and remains associated with its growing rim. This plant-specific mitotic targeting depends on the WPP domain and requires the same amino acids as nuclear-rim association. It is tempting to speculate that plants have acquired a unique targeting domain for RanGAP in connection with its plant-specific mitotic trafficking. ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282 Results Mammalian and plant RanGAP targeting signals are not recognized in the heterologous system The domains responsible for targeting mammalian and plant RanGAP to the NE have no similarity, and there is currently no evidence that the WPP domain is SUMOylated, suggesting that mammalian and plant RanGAP targeting mechanisms differ fundamentally. To rule out that an unrecognized structural similarity exists between the two domains that is recognized by a principally conserved mechanism, we investigated whether the targeting signals of mammalian and plant RanGAP can function in the heterologous system. HeLa cells were transfected with an AtRanGAP1-GFP fusion construct (AtRanGAP1-GFP). Figure 1(a–c) shows that the fusion protein does not accumulate at the HeLa cell NE. When cells were co-transfected with AtRanGAP1-GFP and human RanGAP1 fused to RFP (HsRanGAP1-RFP), and subsequently permeabilized to diffuse soluble proteins from the cytoplasm while retaining proteins tightly associated with the nuclear membrane, AtRanGAP1-GFP is no longer detectable, whereas the HsRanGAP1-RFP signal is retained at the NE (Figure 1d–g). In a parallel experiment using unpermeabilized cells, approximately equal amounts of AtRanGAP1-GFP and HsRanGAP1-RFP fluorescence were detected, demonstrating that both fusion proteins were expressed (data not shown). These data indicate that AtRanGAP1 has no signal for NE association in mammalian cells. To test whether HsRanGAP1 is targeted to the NE in plant cells, GFP-HsRanGAP1 was transiently expressed in Arabidopsis protoplasts and tobacco BY-2 cells. GFP-HsRanGAP1, which is targeted to the nuclear rim in HeLa cells (data not shown), does not accumulate at the nuclear rim of either plant cell type (Figure 1h–k). Together, these data indicate that the WPP domain of AtRanGAP1 and the SUMO-accepting C-terminus of HsRanGAP1 are NE-targeting domains only in their organismal context. Specific point mutations in the WPP domain of AtRanGAP1 abolish NE targeting Currently, only two types of proteins are known to contain WPP domains, plant RanGAP and the plant NE-associated protein MAF1 (Gindullis et al., 1999; Meier, 2000; Patel et al., 2004). We took advantage of the much larger number of available EST sequences for MAF1-like proteins than for plant RanGAPs to identify highly conserved residues in the WPP domain. Figure 2 shows an alignment of known MAFlike and plant RanGAP sequences. Several amino acids are highly conserved among all sequences, including a lowerplant EST from the moss Physcomitrella patens. Mutating 272 Sun Yong Jeong et al. (a) Figure 1. Localization of Arabidopsis and human RanGAP1 in HeLa cells and plant cells. Fusion constructs with GFP or RFP were transiently expressed under the control of the CMV promoter (HeLa cells) or 35S promoter (plant cells). (a) AtRanGAP1-GFP in intact HeLa cell; (b) DNA counterstained with DAPI; (c) overlay of (a) and (b), bar equals 10 lm in a–c. (d) AtRanGAP1-GFP in permeabilized HeLa cell; (e) HsRanGAP1-RFP in permeabilized HeLa cell; (f) DNA counterstained with DAPI; (g) overlay of (d) through (f), bar equals 10 lm in d–g. (h) AtRanGAP1-GFP in tobacco BY-2 cell; (i) GFPHsRanGAP1 in tobacco BY-2 cell, both (h) and (i) counterstained for nucleic acids with SYTO 82 orange. (j) AtRanGAP1-GFP in Arabidopsis protoplast; (k) GFP-HsRanGAP1 in Arabidopsis protoplast. N, nucleus. Bars, 10 lm. (c) (b) (d) (e) (f) (g) (h) (i) (j) (k) N the conserved tryptophan–proline motif in AtRanGAP1 abolishes NE targeting in tobacco BY-2 cells (Rose and Meier, 2001). To investigate the role of other conserved amino acid positions in NE targeting, a series of mutations were introduced into the AtRanGAP1 WPP domain, replacing the wild-type residues with alanine or, in case of alanine, with serine (Figure 2). Mutated proteins fused to GFP were tested for NE targeting in transiently transformed Arabidopsis protoplasts (Figure 3). Mutant mu2 (W18A, P19A) fails to concentrate around the nuclear rim, in agreement with previous observations in tobacco BY-2 cells (Rose and Meier, 2001). Mutants mu3 (T24A, R25A), mu6 (I61A, E62A), and mu7 (Y88A) also abolish NE targeting. In contrast, mu1, mu4, mu5, mu8, mu9, mu11, and mu12 had no effect on NE targeting, indicating that these residues, while conserved, are not required for association of AtRanGAP1 with the NE. AtRanGAP1 associates with the cell plate during mitosis It has been previously shown that the tobacco antigens of an anti-AtRanGAP1 antibody are localized in the area of the spindle and phragmoplast microtubules in dividing cells (Pay et al., 2002). We wished to determine more precisely the subcellular location of AtRanGAP1 during the different stages of the cell cycle. BY-2 cell lines stably expressing AtRanGAP1-GFP were synchronized, counterstained with SYTO 82 orange, and imaged by confocal microscopy. Stages of cell cycle were determined visually, using such landmarks as chromosome condensation, chromosome alignment in the metaphase plate, chromosome separation, N and reappearance of AtRanGAP1-GFP at the NE. Figure 4 shows a series of confocal images representing different stages of cell division and comparing the localization pattern of AtRanGAP1-GFP (Figure 4a) with that of free GFP (Figure 4b). In interphase cells, a strong association of AtRanGAP1-GFP with the NE was observed, while free GFP was equally distributed between the cytoplasm and the nucleus. During metaphase, a concentration in the area of the spindle was observed for AtRanGAP1-GFP, consistent with the data shown by Pay et al. (2002) (compare Figure 5a). However, a similar concentration was also observed with free GFP, indicating that a higher cytoplasmic density exists in the area of the spindle, which might obscure a true spindle association. At late anaphase/early telophase, a strong association of AtRanGAP1-GFP with the position of the cell plate was found. At that stage, free GFP was most prominent in the area of the separating chromosomes and the cytoplasm between the chromosomes (compare Figure 4a, panels g–i, and Figure 4b, panels g–i). At late telophase/early cytokinesis, the most prominent accumulation of AtRanGAP1-GFP was at the rim of the growing cell plate (Figure 4a, panels j - l, and Video 1). At the same stage, AtRanGAP1-GFP reappeared at the nuclear rim, consistent with the reassembly of the NE. At comparable stages, free GFP appeared most concentrated at the chromosomes and in the cytoplasm between the daughter nuclei (Figure 4b, panels j–l). At the end of cytokinesis, once the cell plate has fused with the plasma membrane, the most prominent accumulation of AtRanGAP1-GFP was again with the NEs of the daughter cells (Figure 4a, panels m–o). ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282 Arabidopsis RanGAP1 at the cell plate 273 Figure 2. Sequence conservation of plant WPP domains and residues chosen for mutagenesis. From top: consensus strength given by height of bars, and consensus sequence. Alignment of WPP domain sequences; gray shading indicates amino acids that match the consensus. Abbreviations for species names and GenBank accession numbers are given at the end of each line. A.t., Arabidopsis thaliana, thale-cress; B.v., Beta vulgaris, sugar beet; C.a., Capsicum annuum, jalapeno pepper; C.e., Canna edulis, arrowroot; G.a., Gossypium arboreum, cotton; G.m., Glycine max, soybean; H.c., Hedyotis centranthoides; H.v., Hordeum vulgare, barley; I.n., Ipomoea nil, morning glory; L.e., Lycopersicon esculentum, tomato; L.s., Lactuca sativa, lettuce; L.j., Lotus japonicus, lotus; M.s., Medicago sativa, alfalfa; M.t., Medicago trunculata, barrel medic; M.c., Mesembryanthemum crystallinum, ice plant; O.s., Oryza sativa, rice; P.b., Populus balsamifera, poplar; P.p., Prunus persica, peach; S.t., Solanum tuberosum, potato; Z.m., Zea mays, maize; P. patens, Physcomitrella patens; a, see Meier (2000); b, see Gindullis et al. (1999). Bottom: location of point mutations introduced in AtRanGAP1. , presence/absence of nuclear envelope targeting of mutant AtRanGAP1-GFP fusions. Figure 5 shows images collected after fixing the AtRanGAP1-GFP-expressing, synchronized BY-2 cells. Under these conditions, a larger number of cells at different mitotic stages could be imaged. In addition, the cytoplasmic background of SYTO 82 orange staining due to association with the organelles was strongly reduced. As early as anaphase, a clear accumulation of signal was detected at the position of the cell plate (Figure 5c). The signal grew outward consistent with the location of the growing cell plate (Figure 5d–g). Figure 5(f,g) shows initial accumulation of AtRanGAP1-GFP around the decondensing chromatin (arrows). Figure 5(h) shows a stage comparable to the live image in Figure 4(a, panel j), with accumulation of the fusion protein at the nuclear rim and the outer edges of the cell plate. Figure 6 shows series of optical sections for 3-D reconstructions of the GFP signals in live cells at different stages of cell plate formation. The early stage of cell plate formation corresponding to Figure 4(a, panels g–i) shows a disk-shaped AtRanGAP1-GFP localization (Figure 6a and Video 2). Figure 6(b) (corresponding to Figure 4a, panels ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282 j–l) shows the ring-shaped AtRanGAP1-GFP localization at the later stage of cell plate formation (Video 3). This ringshaped localization pattern of AtRanGAP1-GFP at the late stage of cell plate formation is indistinguishable from the localization of the cell plate marker GFP-DRP2A (Hong et al., 2003) at the same stage (Figure 6c and Video 4). To confirm that the GFP fusion protein properly reflects the localization of AtRanGAP1, an antibody was raised against recombinant AtRanGAP1 and used in immunofluorescence experiments with root tips of 2–3-day-old Arabidopsis seedlings. Figure 7(a) shows that the antibody recognized a single band of the correct size in a total protein extract from WT Arabidopsis seedlings as well as the AtRanGAP1-GFP fusion protein in an extract from a BY-2 cell line expressing a 35S-AtRanGAP1-GFP construct. Figure 7(b) shows double labeling with the AtRanGAP1 antibody (FITC, green) and propidium iodide (red) in an Arabidopsis root tip, demonstrating that endogenous AtRanGAP1 is associated with the nuclear rim in Arabidopsis root cells. Figure 7(c) demonstrates its 274 Sun Yong Jeong et al. (a) (b) N (c) N wt (e) N mu1 mu2 (f) (g) Figure 3. Localization of Arabidopsis RanGAP1 mutants in Arabidopsis protoplasts. AtRanGAP1GFP with introduced point mutations was transiently expressed in Arabidopsis protoplasts and imaged by confocal microscopy. wt, wildtype; mu1–mu12, point mutations at the corresponding amino acid positions, as indicated in Figure 2. Optical sections corresponding to a central plane through the nucleus are shown. N, nucleus. Bars, 10 lm. (d) N mu3 (h) N N N mu4 mu5 (i) (j) N mu6 (k) N mu7 N (l) N N mu8 mu9 mu11 mu12 green (Figure 7d,f) and red (Figure 7e,g) channels of a single cell shown in Figure 7(d,e) and the cell shown in Figure 7(c,f,g). association with the cell plate of a dividing root cell, consistent with the data obtained with the GFP fusion protein. Figure 7(d–g) are separated images from the (b) (a) GFP SYTO a b c d e f g j m h k n GFP Merge Merge SYTO a b c d e f g h i j k l m n o i l o Figure 4. Localization of AtRanGAP1-GFP during cell cycle in live tobacco BY-2 cells. (a) Confocal images of BY-2 cells expressing AtRanGAP1-GFP. (b) Confocal images of BY-2 cells expressing free GFP. GFP, green channel; SYTO, red channel (SYTO 82 orange nucleic acid stain). a–c, interphase; d–f, metaphase; g–i, telophase to early cytokinesis; j–l, cytokinesis; m–o, after completion of cytokinesis. Bars, 10 lm. For a Z-scan through dividing cells expressing AtRanGAP1-GFP, see Video 1. ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282 Arabidopsis RanGAP1 at the cell plate 275 Figure 5. Localization of AtRanGAP1-GFP during cell cycle in fixed tobacco BY-2 cells. (a, b) metaphase; (c) anaphase; (d, e) telophase; (f, g) late telophase, early cytokinesis; (h) late cytokinesis. Green fluorescence, GFP; red fluorescence, SYTO 82 orange nucleic acid stain. The arrows in (c) indicate the association of AtRanGAP1-GFP with the position of the cell plate as early as anaphase. The arrowheads in (f) and (g) point at the initial association of AtRanGAP1GFP with the re-forming nuclear envelope. The arrows in (h) label the outward growing association of AtRanGAP1-GFP with the rim of the cell plate (see also Figure 6) and the arrowheads in (h) indicate the association of AtRanGAP1GFP with the newly formed nuclear envelope. The WPP domain is necessary and sufficient for mitotic targeting of AtRanGAP1 SUMO-1 conjugation is not only essential for targeting HsRanGAP1 to the nuclear pores, but is also required for the association of HsRanGAP with mitotic spindles and kinetochores (Joseph et al., 2002). In addition, RanBP2 co-localizes with HsRanGAP1 throughout the cell cycle, indicating that the same mechanism is involved in targeting HsRanGAP1 in ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282 interphase and during mitosis. Hence, we were interested to determine whether the same (plant-specific) signals are also required to target plant RanGAP1 to its interphase and mitotic locations. To test whether the WPP domain is involved in mitotic targeting of AtRanGAP1-GFP, deletion constructs were fused to GFP and their localization in stably transformed BY-2 cells was investigated. It has been previously shown that the N-terminal 115 amino acids of AtRanGAP1 are necessary and sufficient for targeting to the NE (Rose and Meier, 2001). Figure 8(a) shows that this GFP fusion protein (AtRanGAP1DC-GFP) is robustly targeted to the nuclear rim in interphase cells (panels a–c). During metaphase, fluorescence distribution was similar to AtRanGAP1-GFP (panels d–f). During late anaphase/early telophase a strong association with the cell plate was observed (panels g–i), indistinguishable from the localization pattern of AtRanGAP1-GFP (compare Figure 4). In late telophase/early cytokinesis AtRanGAP1DC-GFP accumulates at the rim of the growing cell plate and reappears at the rims of the daughter nuclei (panels j–l). After the new cell plate has fused with the plasma membrane, the strongest GFP fluorescence was again associated with the NE (panels m–o). In contrast, AtRanGAP1DN-GFP was not found specifically associated with any cellular structure throughout the cell cycle (Figure 8b). Somewhat higher fluorescence intensity was observed in the cytoplasm accumulated around the rim of the growing cell plate in telophase (Figure 8b, panels g–i). However, the same accumulation was observed for free GFP, indicating a general abundance of cytoplasmic proteins in this area (compare Figure 8b, panels j–l). To confirm that the same localization signal also targets AtRanGAP1-GFP to the NE and the cell plate in Arabidopsis plants, transgenic lines were created which expressed either AtRanGAP1-GFP or AtRanGAP1DC-GFP under the control of the 35S promoter. Root tips from 2–3-day-old seedlings were imaged for GFP fluorescence. Figure 8(c) shows that AtRanGAP1-GFP is targeted to the NE in interphase root cells and to the cell plate in dividing root cells. AtRanGAP1DC-GFP showed the same localization pattern as AtRanGAP1-GFP, demonstrating that the WPP domain is sufficient for interphase and mitotic targeting in Arabidopsis plants as well (Figure 8d). Specific point mutations disrupt mitotic targeting of AtRanGAP1 We wanted to investigate whether the same point mutations leading to disruption of NE targeting would also disrupt cellplate association of AtRanGAP1. Three point mutations were selected that had shown no association with the NE in transient assays (mu3, mu6, and mu7, see Figure 2). All three point mutations showed no specific targeting throughout cell division, behaving indistinguishably from 276 Sun Yong Jeong et al. Figure 6. AtRanGAP1-GFP concentrates at the newly forming cell plate in dividing tobacco BY2 cells. (a) Three-dimensional reconstruction of diskshaped AtRanGAP1-GFP localization at the early stage of cell plate formation, rotated around the vertical axis by 10 degrees for each image (for rotation movie, see Video 2). (b) Three-dimensional reconstruction of ringshaped AtRanGAP1-GFP localization at the later stage of cell plate formation, rotated around the horizontal axis by 10 degrees for each image (for rotation movie, see Video 3). (c) Three-dimensional reconstruction of cellplate marker GFP-DRP2A localization at the later stage of cell plate formation, rotated around the vertical axis by 10 for each image (for rotation movie, see Video 4). the WPP domain deletion mutant AtRanGAP1DC-GFP (Figure 9 and Figures S1 and S2). This indicates that the threonine–arginine pair in position 24 and 25, the isoleucine–glutamate pair in positions 61 and 62, and the tyrosine in position 88 are required for targeting AtRanGAP1 both to the NE and the cell plate and suggests that – as in human cells – a common mechanism is involved in these different targeting events of plant RanGAP throughout the cell cycle. Discussion Diverse RanGAP targeting in different kingdoms Based on localization and the presence or absence of targeting domains, at least three classes of eukaryotic RanGAPs can be divided. The simplest form is represented by yeast RanGAP, which has no apparent signals for targeting to the nuclear rim or to mitotic structures, and appears to remain cytoplasmic throughout the cell cycle (Hopper et al., 1990). Yeast RanGAP consists of a leucine-rich repeat and an acidic domain, which are both required for RanGAP activity (Hillig et al., 1999; Melchior et al., 1993). Both domains are conserved in plant and vertebrate RanGAP, but are accompanied by kingdom-specific domains necessary to anchor RanGAP to the outer surface of the NE. We have shown here that the plant and mammalian RanGAP targeting domains are not functional in the heterologous cell system. No consensus sequence for SUMOylation is present on the WPP domain and no higher molecular weight isoforms of AtRanGAP1 have been observed in immunoblots (e.g., Figure 7a), suggesting that, unlike for human RanGAP, SUMOylation is probably not involved in subcellular targeting of plant RanGAP. Together with the apparent lack of a plant RanBP2 homolog, this is consistent with the hypothesis that RanGAP is targeted by a different mechanism to the nuclear rim in plants. Plant RanGAP has specific mitotic locations There are a number of differences between animal and plant mitosis, one of the most prominent being the way in which the new membrane division between the two daughter cells is established. Cytokinesis in plants does not involve contraction and fusion of a cleavage furrow. ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282 Arabidopsis RanGAP1 at the cell plate 277 Figure 7. Immunofluorescence microscopy of Arabidopsis root tip cells. (a) The AtRanGAP1 antibody detects a single band of ca. 60 kDa in a total protein extract from Arabidopsis seedlings (lane 1, arrow) and a single band of ca. 80 kDa in a total protein extract of the AtRanGAP1-GFP BY-2 cell line (lane 2, arrow). Asterisks mark a weak unspecific signal seen in all plant protein extracts tested (lanes 1 and 2 and data not shown). (b) The AtRanGAP1 antibody decorates the nuclear envelope in root tip cells. Green fluorescence, FITC; red fluorescence, propidium iodide counterstaining of nuclei. (c) The AtRanGAP1 antibody decorates a newly forming cell plate in an Arabidopsis root tip cell. Green, FITC; red, propidium iodide. (d, e) Grayscale images of split channels of a single nucleus shown in (b). (d) green channel; (e) red channel. (f, g) Grayscale images of split channels of the dividing cell shown in (c). (f) Green channel; (g) red channel. Arrowheads in (c) and (f) indicate the cell plate. Instead, a new double membrane is assembled from vesicles, which migrate along the microtubules of the phragmoplast toward the equatorial plane where they fuse to form the cell plate, which grows outward to eventually fuse with the plasma membrane (Bednarek and Falbel, 2002; Hong et al., 2001). Like mammalian RanGAP, plant RanGAP goes through a specific pattern of re-localization during cell cycle. Tobacco RanGAP is localized in the area of the spindle and the phragmoplast in dividing cells (Pay et al., 2002). We have shown here in addition that AtRanGAP1 is targeted to the cell plate during cytokinesis and that the WPP domain is necessary and sufficient for this targeting. During metaphase, we have observed an accumulation of AtRanGAP1GFP in the vicinity of the spindle as well (e.g., see Figure 4a, panel d–f and Figure 5a–c). However, our assay makes it difficult to distinguish between true spindle association and accumulation of the fusion protein in the dense cytoplasm that surrounds the spindle because free GFP also accumulates in this area of the cell (Figure 4b, panels d–f). We therefore consider it is likely that AtRanGAP1 is associated both with the vicinity of the spindle in metaphase as reported by Pay et al. (2002) and with the cell plate during cytokinesis as described here. Indeed, performing a microtubule precipitation experiment as described by Pay et al. (2002), we also found that AtRanGAP1-GFP is found in the pellet. Interestingly, the point mutant mu3, which disrupted association of AtRanGAP1-GFP with the NE and the cell plate, did not alter the association of the fusion protein with the microtubule pellet. In contrast, deletion of the C-terminus of AtRanGAP1 made the remaining WPP-domain-GFP fusion protein (AtRanGAP1DC-GFP) partition with the supernatant (data not shown). These findings suggest that the association of plant RanGAP with microtubules might be a ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282 feature that is independent of its targeting to the membrane structures. Association of AtRanGAP1 with the cell plate was observed as early as anaphase (Figure 5). This is consistent with the earliest accumulation of vesicles in the area of the future cell plate (Segui-Simarro et al., 2004). Formation of the cell plate involves the fusion of vesicles, which starts at the center of the cell plate and expands centrifugally outward as the area of the cell plate enlarges. During later stages of cell plate growth, microtubules depolymerize at the center of the cell plate and new ones form around the margins where new vesicles are delivered (Segui-Simarro et al., 2004). The localization of RanGAP1 resembles the area of the cell plate that undergoes vesicle fusion and closely resembles that of dynamin. Strikingly, tomato MAF1, a representative of the only other plant protein family containing a WPP domain, also migrates from the NE to the cell plate during cytokinesis (Patel et al., 2004). The Arabidopsis homologs of MAF1, WPP1 and WPP2 are also associated with the NE, indicating that the WPP domain provides information for both NE and cell plate localization in plants. Vertebrate RanGAP is required for the fusion of the membrane vesicles that assemble on the decondensing chromatin and form the new NEs (Hetzer et al., 2000; Zhang and Clarke, 2000; Zhang et al., 2002). While nothing is currently known about mitotic roles of the Ran cycle in plants, it is interesting to note that the mitotic position of AtRanGAP1 at the growing edge of the cell plate also correlates with a membrane fusion event. There are clearly differences in mitotic membrane trafficking between the kingdoms. Plant Golgi stacks do not vesiculate and the process of secretion and vesicle transport remains active during plant mitosis (Bednarek and Falbel, 2002; Nebenführ et al., 2000). Both Golgi vesicles and tubular ER components 278 Sun Yong Jeong et al. (a) (b) GFP SYTO a b c d e f g h i j m k n GFP Merge SYTO Merge a b c d e f g h i j k l m n o l o (c) (d) Figure 8. The WPP domain is necessary and sufficient for AtRanGAP1-GFP localization during cell cycle. (a) Confocal images of BY-2 cells expressing AtRanGAP1DC-GFP (WPP domain). (b) Confocal images of BY-2 cells expressing AtRanGAP1DN-GFP (WPP domain deletion). GFP, green channel; SYTO, red channel (SYTO 82 orange nucleic acid stain). a–c, interphase; d–f, metaphase; g–i, telophase to early cytokinesis; j–l, cytokinesis; m–o, after completion of cytokinesis. Bars, 10 lm. (c) AtRanGAP1-GFP localization in primary root tip cells of transgenic Arabidopsis plants. Arrows indicate the cell plate of a dividing cell. (d) AtRanGAP1DC-GFP localization in primary root tips of transgenic Arabidopsis plants. Arrows indicate the cell plate of a newly divided cell. are recruited to the division plane during mitosis and cytokinesis (Cutler and Ehrhardt, 2002). Members of the Arabidopsis Rab family have demonstrated roles in intracellular membrane trafficking and it was hypothesized that Rab GTPases would, in conjunction with SNARE proteins, provide specificity for membrane fusion events (for reviews, see Stenmark and Olkkonen, 2001; Zerial and McBride, 2001). The data presented here provide an opening into investigating whether the Ran cycle, too, has a role in the formation of the cell plate. The WPP domain is required for mitotic RanGAP targeting in plants We have proposed previously that both in vertebrates and plants the subcellular targeting of RanGAP is more relevant ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282 Arabidopsis RanGAP1 at the cell plate 279 GFP SYTO Merge a b c d e f membrane re-organization events discussed here (NE breakdown and reformation; phragmoplast-type cell plate formation) occur together in many algal taxa that are basal to the lineage of the land plants, such as Coleochaetales (Marchant and Pickett-Heaps, 1973), Charales (Pickett-Heaps, 1967), and Zygnematales (Fowke and Pickett-Heaps, 1969a,b; LopezBautista et al., 2003). Therefore, an unrecognized mechanistic connection might exist in plants between these two membrane rearrangement events. Experimental procedures g h i j k l m n o Figure 9. Point mutations in AtRanGAP1-GFP abolish targeting during cell cycle. Confocal images of BY-2 cells expressing AtRanGAP1-GFP with a point mutation at position 3 (see Figure 2 and Experimental procedures). GFP, green channel; SYTO, red channel (SYTO 82 orange nucleic acid stain). a–c, interphase; d–f, metaphase; g–i, telophase to early cytokinesis; j–l, cytokinesis; m–o, after completion of cytokinesis. Bars, 10 lm. For corresponding Figures of AtRanGAP1-GFP with point mutation at positions 6 and 7, see Figures S1 and S2. for its function in mitosis than for its association with the nuclear pore in interphase (Joseph et al., 2002; Rose and Meier, 2001). This is based on the correlation of specific RanGAP targeting domains with the occurrence of open mitosis in higher eukaryotes. SUMO modification appears to be confined to metazoan RanGAP and is involved in the process that spatially regulates the RanGAP position during the cell cycle (Joseph et al., 2002). We have shown here that the different subcellular addresses of plant RanGAP during interphase and cell cycle also require the same signal sequence. However, this signal fundamentally differs from the one utilized by vertebrate RanGAP. The use of a different signal correlates with the unique, plant-specific positioning during cytokinesis at the cell plate. It is conceivable that the specific targeting mechanism of higher plant RanGAP has evolved in parallel with the occurrence of phragmoplast-type open mitosis. Plant and animal RanGAP might therefore have acquired different targeting domains binding to different protein interaction partners suitable for their subcellular trafficking ‘needs.’ Consistent with this hypothesis we have identified a colocalizing interaction partner of the WPP domain, which is a protein unique to plants (unpublished data). Indeed, the two ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282 Constructs pRTL-AtRanGAP1-GFP (Rose and Meier, 2001) and pDsRed-hRanGAP1 (Joseph et al., 2002) were described previously. For cloning of the AtRanGAP1 coding sequence into a mammalian expression vector, the NcoI fragment was filled in and ligated into the SmaI site of pEGFP-N2 (Clontech, Palo Alto, CA, USA) to obtain pEGFPAtRanGAP1. This provides expression of AtRanGAP1 as a fusion protein with GFP at its C-terminus. For cloning of the HsRanGAP1 coding sequence into a plant expression vector, the coding sequence was PCR-amplified using the primers 5¢-GAAGATCTATGGCCTCGGAAGACATTG-3¢ and 5¢-GAAGATCTGACCTTGTACAGCGTCTGC3¢ and cloned into the BglII site of pRTL2-mGFPS65T (von Arnim et al., 1998) using the internal BglII sites of the PCR primers to obtain pRTL-HsRanGAP1-GFP. This provides expression of HsRanGAP1 as a fusion protein with GFP at its N-terminus. For stable BY-2 cell transformation, the fragments of AtRanGAP1-GFP, AtRanGAP1DCGFP, AtRanGAP1DN-GFP (Rose and Meier, 2001), AtRanGAP1 (mu3, mu6 and mu7)-GFP (see below) and free GFP (mGFPS65T) were digested with XhoI and partially with XbaI (RanGAP1 has an internal XbaI site) and cloned into XhoI and SpeI in the binary vector pFGC1008 (http://www.arabidopsis.org). The same constructs were used to transform Arabidopsis plants. Site-directed mutagenesis Point mutations were introduced into AtRanGAP1-GFP as described previously (Rose and Meier, 2001) using the following mutagenic primers and their respective reverse primers (introduced nucleotide changes are underlined) – mu1: 5¢-CAGAACCGTGTTTTGGCAGTGAAGATGTGG-3¢; mu2: 5¢-GTCAGTGAAGATGGCGGCACCGAGTAAGAG-3¢; mu3: 5¢-CGAGTAAGAGTGCCGCTCTCATGCTTGTTG-3¢; mu4: 5¢-CTCATGCTTGTTGAGGCGATGACCAAGAAC-3¢; mu5: 5¢-CTTCTCCAGGAAGGCCGCTCTTTTGTCTG-3¢; mu6: 5¢-CGCCAAGCGCGCTGCAGATTTGGCCTTTG-3¢; mu7: 5¢-CTGCTGTTCACGTCGCTGCTAAAGAATCC-3¢; mu8: 5¢-CACGTCTATGCTAAAGCATCCAGCAAGCTC-3¢; mu9: 5¢-CTATGCTAAAGAATCCGCCAAGCTCATGCTTG-3¢; mu11: 5¢-GTCTGTTGAAGAGTCTGAGCAAGACGCCAAG-3¢; mu12: 5¢-GGCTGAGCAAGACTCCAAGCGCATTGAAG-3¢. The mutations were designed to replace residues in the wild-type sequence with either alanine (mu1–9) or serine (mu11 and 12) and were confirmed by sequencing. Mammalian cell culture, transfection and fluorescence microscopy HeLa cells were cultured in DMEM medium (BioFluids, Rockville, MD, USA) supplemented with 10% FBS (Gemini-bioproducts, 280 Sun Yong Jeong et al. Calabasas, CA, USA) at 37C in a humidified incubator with 5% CO2. Cells grown on cover slips were co-transfected with pEGFP-AtRanGAP1 and pDsRed-hRanGAP1 using Effectene Transfection Reagent (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. Cells were fixed with 3.7% paraformaldehyde in PBS for 20 min (Figures 1a–c) or permeabilized with 0.005% digitonin (SigmaAldrich, St Louis, MO, USA) in transport buffer [110 mM KOAc, 20 mM HEPES, pH 7.3, 2 mM Mg(OAc)2, 0.5 mM EGTA, 2 mM DTT, 1 lg ml)1 each of leupeptin, pepstatin, and aprotinin] for 5 min and fixed with 3.7% paraformaldehyde (Figure 1d–g) 48 h after transfection. Samples were stained with DAPI for visualizing DNA and mounted in Vactashield anti-fade mounting medium (Vector Laboratories, Burlingame, CA, USA). Slides were examined with a Zeiss Axioskop fluorescence microscope (Carl Zeiss Inc., Thornwood, NY, USA) and images were collected and analyzed with Openlab software. Plants and plant cell cultures Arabidopsis plants (Jeong et al., 2003) and tobacco BY-2 suspension culture cells (Rose and Meier, 2001) were cultured as described previously. Green Arabidopsis suspension culture cells were cultured in Gamborg’s B-5 Basal medium with minimal organics (Caisson Laboratories, Rexburg, ID, USA) supplemented with 2% (w/v) sucrose, 5 lM 2,4-dichloro-phenoxyacetid acid, and 0.05% (w/v) MES, pH 5.7. Cultures were maintained by shaking at 200 rpm in constant light at 24C and subcultured weekly by 1:10 dilution with fresh medium. Arabidopsis protoplast transformation Four- to five-day-old Arabidopsis suspension culture cells were harvested by centrifugation for 10 min at 300 g, washed once with the same volume (0.4 M mannitol/20 mM MES, pH 5.5), and incubated at room temperature on a platform shaker at 150 rpm in 1% cellulase, 0.1% pectolyase (Karlan, Santa Rosa, CA, USA) in the same volume (0.4 M mannitol/20 mM MES, pH 5.5). After completion of protoplast formation (2–5 h, monitored via microscopy), protoplasts were harvested through centrifugation for 5 min at 150 g, washed once with 1/2 volume ice-cold W5 medium (154 mM NaCl, 5 mM KCl, 125 mM CaCl2, 5 mM glucose, pH 6), resuspended in ice-cold W5 medium at a concentration of 3–5 · 106 cells ml)1, and incubated on ice for 2 h. Immediately prior to transformation, the protoplasts were harvested at 4C and resuspended in ice-cold MaMg solution (0.4 M mannitol, 15 mM MgCl2, 5 mM MES, pH 5.6). 20 lg DNA, 300 ll protoplast suspension and 300 ll of 40% PEG6000 (Calbiochem, La Jolla, CA, USA) in 0.1 M Ca(NO3)2, 0.4 M mannitol, pH 8.0, were mixed by gentle inversion at room temperature for 30 min. The suspension was slowly diluted with 8 ml W5 medium and the protoplasts harvested via centrifugation for 5 min at 50 g. After removal of the supernatant, the transformed protoplasts were resuspended in 4 ml Gamborg’s medium containing 0.4 M mannitol and incubated in the dark for 48 h prior to microscopy. BY-2 cell transformation Transient transformation of BY-2 cells was performed using a biolistic DNA delivery method essentially as described previously for NT-1 cells (Gindullis et al., 1999). For stable transformation of BY-2 cells, all constructs of AtRanGAP1-GFP, AtRanGAP1DC-GFP, AtRanGAP1DN-GFP, AtRanGAP1 (mu3, mu6 and mu7)-GFP and free GFP (mGFPS65T) in pFGC1008 were transformed into Agrobacterium tumefaciens (strain LBA4404) and colonies were selected with 8.5 lg ml)1 chloramphenicol and 50 lg ml)1 streptomycin. Agrobacterium-mediated transformation of BY-2 cells was performed as described by Hong et al. (2001). Transformed calli were selected on MS agar plates containing 25 lg ml)1 of hygromycin and 250 lg ml)1 of carbenicilline after 4 weeks. The selected calli were transferred to new MS agar plates containing the same concentration of antibiotics as above. After 1 week, calli were transferred into MS liquid media containing 20 lg ml)1 of hygromycin to establish suspension cultures. BY-2 cell synchronization Synchronization of BY-2 cells was performed essentially as described by Nagata and Kumagai (1999) with the following modifications. Seven-day-old BY-2 cells were transferred to 45 ml of fresh MS medium and 5 lg ml)1 of aphidicolin (Sigma-Aldrich) in DMSO was added. Twenty-four hours after incubation in the dark, cells were collected by filtering through sterile miracloth and washed with 200 ml of fresh MS medium. Cells were transferred into 100 ml of fresh MS medium and were continuously cultured. About 50% of M-phase cells, about 30% of late G2 phase and about 10% of G1 phase could be identified after 10 h release from aphidicolin. For improved synchronization, 5 h after release from aphidicolin, 50 ml of the culture was transferred to a new flask and 1.54 lg ml)1 of propyzamide (Sigma-Aldrich) in DMSO was added. After 4 h of continued culture in the presence of propyzamide, cells were washed as described above and transferred to fresh medium. Two hours after release from propyzamide, more than 60% of M-phase cells, about 20% of late G2 phase and about 10% of G1 phase were typically detected. Confocal microscopy of plant cells SYTO 82 orange (Molecular Probes, Eugene, OR, USA) was used to stain nucleic acids as described previously (Rose and Meier, 2001), using either 500 nM SYTO 82 for 45 min or 1 lM of SYTO 82 for 5–10 min. Digitized confocal images were acquired on a PCM2000/ Nikon Eclipse E600 confocal laser scanning microscope as previously described (Rose and Meier, 2001). For preparing Z-series of images and 3-D movies, the software ‘Simple PCI’ (Compix Imaging System, Cranberry Township, PA, USA) was used. For Z-series, images were scanned in 0.75 lm interval optical sections using a 40X objective lens and 2X rolling average image processing. For 3-D movies, optical sections were stacked with a setting of 0.3 lm for the pixel size (X–Y) and 0.75 lm for the step size (Z). The image field (X–Y) and range of optical sections (Z) were chosen for the best representation of the cell plates. Immunofluorescence microscopy Fixation of BY-2 cells and Arabidopsis root tips, and immunolabeling of Arabidopsis root tips were carried out essentially as described by Woo et al. (1999) and Smertenko et al. (2004) with the following modifications. To fix BY-2 cells expressing AtRanGAP1GFP, synchronized cells were incubated with a fixative containing 3.7% of paraformaldehyde, 0.2% picric acid and 5 mM EGTA in PBS buffer for 1 h at room temperature and the fixative was washed out with PBS three times for 10 min each. The cells were kept in PBS at 4C before observation of GFP fluorescence. Two-day-old Arabidopsis seedlings were grown in MS liquid media by shaking at 200 rpm in constant light at 24C. Seedlings ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282 Arabidopsis RanGAP1 at the cell plate 281 were fixed for 30 min at room temperature in 4% paraformaldehyde in 0.1 Pipes, pH 6.8, 5 mM EGTA, 2 mM MgCl2 and 0.4% Triton X-100. The fixative was removed by three 10-min washes in PBS. Cells were treated with 1% cellulase, 0.1% pectolyase (Karlan), 0.4 M mannitol, 20 mM MES, 5 mM EGTA, 1 mM PMSF and 5 ll ml)1 of protease inhibitor cocktail (Sigma-Aldrich) for 20 min to digest cell walls. Cells were washed three times for 10 min each with PBS, attached to polyL-Lys-coated slides and semi-dried. Cells were permeabilized with 1% Triton X-100 in PBS for 10 min and washed three times for 10 min each. Blocking was carried out with 0.05% Tween-20, 1% BSA in PBS for 1 h. Cells were labeled with the primary antibody (rabbit polyclonal anti-AtRanGAP1) diluted 1:100 in PBS containing 0.05% Tween-20 and 1% BSA overnight at 4C. The specimens were then washed three times for 10 min each with PBS and incubated for 1 h at room temperature with FITC-conjugated sheep anti-rabbit secondary antibody (Sigma-Aldrich) diluted 1:200 in PBS containing 0.05% Tween-20 and 1% BSA. The specimens were washed with PBS two times for 20 min and incubated with 10 lg ml)1 of propidium iodide in water for 10 min and washed with PBS two times for 20 min. The specimens were mounted with mounting medium (Sigma-Aldrich) and the slides were placed in the dark for at least 1 h before observation under the confocal microscope. Immunoblot analysis Immunoblot analysis was performed as described in Jeong et al. (2003). The anti-AtRanGAP1 antibody was used at a 1:5000 dilution and anti-GFP rabbit polyclonal (Molecular Probes) was used at a 1:1500 dilution. Acknowledgements We greatly acknowledge the help by Dr Tomasz Calikowski and Xianfeng Xu in developing the AtRanGAP1 antibody. We would like to thank Dr Desh Pal S. Verma and Dr Zonglie Hong for the Arabidopsis cell suspension culture and for providing a BY-2 cell line expressing GFP-DRP2A. We thank Dr Biao Ding for generous user time of his confocal microscope. Financial support by the National Science Foundation (MCB-0079577, MCB-0343167, and MCB209339) and the US Department of Agriculture (Plant Growth and Development 2001-01901) to I.M. is greatly acknowledged. Supplementary Material The following material is available from http://www. blackwellpublishing.com/products/journals/suppmat/TPJ/TPJ2368/ TPJ2368sm.htm Video 1 (1.6 MB). Scan through BY-2 cells stably expressing AtRanGAP1-GFP (green), counterstained for nucleic acids with SYTO 82 orange (red). The center cell is in early cytokinesis (note the RanGAP1-GFP signal at the growing cell plate), the cells on the left and right are in late cytokinesis and show the typical interphase pattern of AtRanGAP1-GFP concentration at the NE. Video 2 (2.5 MB). Rotation of a 3-D reconstruction of the cell plate in early cytokinesis of a BY-2 cell stably expressing AtRanGAP1-GFP. Video 3 (2.8 MB). Rotation of a 3-D reconstruction of the cell plate in late cytokinesis of a BY-2 cell stably expressing AtRanGAP1-GFP. Video 4 (2.3 MB). Rotation of a 3-D reconstruction of the cell plate in late cytokinesis of a BY-2 cell stably expressing GFP-DRP2A. Figure S1. Point mutations in AtRanGAP1-GFP abolish targeting during cell cycle. Confocal images of BY-2 cells expressing AtRanGAP1-GFP with a point mutation at position 6 (see Figure 2 ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282 and Experimental procedures). GFP, green channel; SYTO, red channel (SYTO 82 orange nucleic acid stain). a–c, interphase; d–f, metaphase; g–i, telophase to early cytokinesis; j–l, cytokinesis; m–o, after completion of cytokinesis. Bars, 10 lm. Figure S2. Point mutations in AtRanGAP1-GFP abolish targeting during cell cycle. Confocal images of BY-2 cells expressing AtRanGAP1-GFP with a point mutation at position 7 (see Figure 2 and Experimental procedures). 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(1999) Characterization of nuclear import of potato spindle tuber viroid RNA in permeabilized protoplasts. Plant J. 17, 627–635. Zerial, M. and McBride, H. (2001) Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2, 107–117. Zhang, C. and Clarke, P.R. (2000) Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts. Science, 288, 1429–1432. Zhang, C., Hutchins, J.R., Mühlhäusser, P., Kutay, U. and Clarke, P.R. (2002) Role of importin-beta in the control of nuclear envelope assembly by Ran. Curr. Biol. 12, 498–502. ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282